This proposal concerns the c-raf-1 proto-oncogene, which encodes a serine/threonine protein kinase. A large number of studies have implicated the Raf-1 kinase at a central position in mediating the transmission of mitogenic signals initiated by several growth factors. Raf is also involved in mediating responses to a diverse variety of other stimuli, such as inflammatory cytokines, differentiation signals, and UV light. The assumption is that Raf, in turn, regulates the activity of other cellular proteins through phosphorylation. The presence of activated Raf kinase has been associated with several types of human malignancies. Understanding the molecular mechanisms by which cells distinguish and respond to extracellular signals will require the identification of the individual components of the signal transduction pathways. Raf has been shown to mediate the activation of the MAP kinase pathway by phosphorylating MAP kinase kinase (MEK). We have recently implicated the IkappaB inhibitor of the NF-kappaB transcription factor as a substrate of the Raf kinase. The central aim of this proposal is two-fold. The first aim is to perform detailed studies of the Raf-IkappaB relationship to determine whether Raf is a physiological IkappaB kinase, and to investigate the biological significance of IkappaB phosphorylation by Raf. Towards this end we will perform in vivo studies to determine whether Raf can activate NF-kappaB through a MEK/MAPK-independent pathway. We will use in vitro kinase assays with purified components to quantitatively compare IkappaB and MEK as Raf substrates. We will study in vitro the functional consequences of IkappaB phosphorylation in an IkappaB/NF-kappaB complex on activation of NF-kappaB. The second aim is to identify and characterize additional substrates of the Raf kinase. Several lines of evidence indicate that other, perhaps many, Raf substrates remain to be discovered. This will be approached using the yeast two hybrid system to identify proteins that interact with the kinase domain of Raf. Putative candidates will be tested for in vitro and in vivo phosphorylation by Raf kinase. The experiments outlined in this proposal will provide a better understanding of the function of Raf in normal cellular physiology, and ultimately, its oncogenic action.